Increasing the Understanding of Nutrient Transport Capacity of the Ovine Placentome.

Placental nutrient transport capacity influences fetal growth and development; however, it is affected by environmental factors, which are poorly understood. The objective of this study was to understand the impact of the ovine placentome morphological subtype, tissue type, and maternal parenteral supplementation of arginine mono-hydrochloride (Arg) on nutrient transport capacity using a gene expression approach. Placentomal tissues of types A, B, and C morphologic placentome subtypes were derived from 20 twin-bearing ewes, which were infused thrice daily with Arg (n = 9) or saline (Ctrl, n = 11) from 100 to 140 days of gestation. Samples were collected at day 140 of gestation. Expression of 31 genes involved in placental nutrient transport and function was investigated. Differential expression of specific amino acid transporter genes was found in the subtypes, suggesting a potential adaptive response to increase the transport capacity. Placentomal tissues differed in gene expression, highlighting differential transport capacity. Supplementation with Arg was associated with differential expressions of genes involved in amino acid transport and angiogenesis, suggesting a greater nutrient transport capacity. Collectively, these results indicate that the morphological subtype, tissue type, and maternal Arg supplementation can influence placental gene expression, which may be an adaptive response to alter the transport capacity to support fetal growth in sheep.

The role of the oviduct environment in embryo survival.

CONTEXT: Declining fertility is an issue in multiple mammalian species. As the site of fertilisation and early embryo development, the oviduct plays a critical role in embryo survival, yet there is a paucity of information on how the oviduct regulates this process. AIMS: We hypothesised that differences in steroid hormone signalling and/or immune function would be observed in a model of poor embryo survival, the peripubertal ewe. METHODS: We examined expression of steroid hormones in systemic circulation, oviductal expression of oestrogen receptorαand genes important in steroid hormone signalling, and immune function in pregnant and cyclic peripubertal and adult ewes on day 3 after oestrus. KEY RESULTS: Concentrations of progesterone, but not oestradiol, were decreased in the peripubertal ewe compared to the adult ewe. Oestrogen receptorαprotein expression was increased in the peripubertal ewe, but pathway analysis of gene expression revealed downregulation of the oestrogen signalling pathway compared to the adult ewe. Differential expression of several genes involved in immune function between the peripubertal and adult ewe was consistent with an unfavourable oviductal environment in the peripubertal ewe lamb. Oestradiol concentration was positively correlated with the expression of multiple genes involved in the regulation of immune function. CONCLUSIONS: Differences in the immune environment of the oviduct, potentially linked to differential modulation by steroid hormones, may partially underly the poor fertilisation and early embryo survival observed in the peripubertal ewe. IMPLICATIONS: A unfavourable oviductal environment may play an important role in limiting reproductive success.

A novel, stain-free, natural auto-fluorescent signal, Sig M, identified from cytometric and transcriptomic analysis of infectivity of Cryptosporidium hominis and Cryptosporidium parvum.

Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan Cryptosporidium. The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the Cryptosporidium parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective in vitro system for Cryptosporidium culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the C. parvum-specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with C. parvum or C. hominis. The Cryptosporidium parvum-infected cells showed higher levels of signal using Sporo-Glo™ than C. hominis-infected cells, which was likely because Sporo-Glo™ was generated against C. parvum. We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both C. parvum and C. hominis cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing Cryptosporidium infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter® analysis to investigate the transcriptomic landscape for the two Cryptosporidium species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular Cryptosporidium gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to Cryptosporidium infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of Cryptosporidium infectivity.

Effect of birth rank, and placentome subtype on expression of genes involved in placental nutrient transport in sheep.

Placental function is a key determinant of fetal growth and development that can be influenced by maternal and fetal environmental factors. The molecular mechanisms by which the placenta senses and responds to environmental cues are poorly understood. This exploratory study aimed to characterize the effect of birth rank (single vs. twin) and placentome morphologic subtype on expression of genes involved in nutrient transport, angiogenesis, immunity and stress response. Cotyledonary tissue was collected from type A, B and C placentomes from five single and six twin fetuses at 140 days of gestation. GLUT1 and GLUT3 were the most highly expressed genes consistent with the high demand for glucose to support fetal growth. Expression of BCKDHß and IGF-2 was 1.3- and 1.5-fold higher, respectively, and PCYT1A was 3-fold lower in singles compared to twins (P < 0.05) while no other differences in gene expression were observed between birth ranks. Expression of EAAT2 and LAT2 was higher while PCYT1A was lower in A compared to B type cotyledons. Expression of GUCY1B1/3 and IGF-1 was higher while CD98 and LAT2 were lower in type B compared to C cotyledons (P < 0.05). Compared to type C cotyledons, expression of EAAT2, IGF-1, IGF-2, LAT1 was higher, while TEK was lower in type A cotyledons. The effects of birth rank on placental gene expression in this study indicated that placental nutrient transport and/or function differs between single and twin pregnancies in sheep. Differences in gene expression between the placentome subtypes suggests that changes in placentome morphology are associated with shifts in amino acid transport and metabolism, oxidative stress and angiogenesis and/or blood flow. This study highlights that placental gene expression differs in response to birth rank and placentome morphologic subtype which suggests that both maternal and fetal factors may influence placental function in sheep. These associations provide insights into gene pathways for more targeted future investigations as well as potential adaptations to improve placental efficiency to support fetal growth in twin pregnancies.

Cross-reactivity of antibodies to different rumen methanogens demonstrated using immunomagnetic capture technology.

Methane is produced in the rumen of ruminant livestock by methanogens, accounting for approximately 14.5% of anthropogenic greenhouse gas emissions in terms of global warming potential. The rumen contains a diversity of methanogens species, and only a few of these have been cultured. Immunomagnetic capture technology (ICT) is a simple and effective method to capture and concentrate target organisms in samples containing complex microflora. We hypothesized that antibody-coated magnetic beads could be used to demonstrate antibody specificity and cross-reactivity to methanogens in rumen samples. Sheep polyclonal antibodies raised against four isolates of rumen dwelling methanogens, Methanobrevibacter ruminantium strain M1, Methanobrevibacter sp. AbM4, Methanobrevibacter sp. D5, and Methanobrevibacter sp. SM9 or an equal mix of all four isolates, were used to coat paramagnetic beads. ICT was used together with flow cytometry and qPCR to optimize key parameters: the ratio of antibody to beads, coupling time between antibody and paramagnetic beads to produce immunomagnetic beads (IMBs), and optimal incubation time for the capture of methanogen cells by IMBs. Under optimized conditions, IMBs bound strongly to their respective isolates and showed a degree of cross-reactivity with isolates of other Methanobrevibacter spp. in buffer and in rumen fluid, and with resident methanogens in rumen content samples. The evidence provided here indicates that this method can be used to study the interaction of antibodies with antigens of rumen methanogens, to understand antigen cross-reactivity and antibody binding efficiency for the evaluation of antigens used for the development of a broad-spectrum anti-methanogen vaccine for the abatement of methane production.

Growth performance, antibody response, and mammary gland development in New Zealand dairy replacement bovine heifers fed low or high amounts of unpasteurized whole milk.

This study evaluated the influence of feeding low and high preweaning allowances of unpasteurized whole milk (MA) on intake, selected blood metabolites, antibody response, mammary gland growth, and growth of New Zealand (NZ) dairy heifers to 7 mo of age. At 10 ± 2 d of age (study day 0), group-housed (six·pen-1) heifer calves (Holstein-Friesian × Jersey) were allocated to low (4 L whole milk·calf-1·d-1; n = 7 pens) or high (8 L whole milk·calf-1·d-1; n = 7 pens) MA for the next 63 d. Calves were gradually weaned between days 63 ± 2 and 73 ± 2. Calves in each pen had ad-libitum access to clean water, pelleted calf starter, and chopped grass hay from day 1 to 91 ± 2 d. At 92 ± 2 d, all calves were transferred to pasture, grazed in a mob, and their growth and selected blood metabolites were measured until day 209. All animals were weighed weekly during the indoor period (to day 91) and then at days 105, 112, 128, 162, 184, and 209. Skeletal growth measurements and blood samples to analyze selected metabolites were collected at the start of the experiment, weaning, and then postweaning on day 91, and day 201. Specific antibodies against Leptospira and Clostridia were quantified in weeks 7, 13, and 27. Mammary glands were scanned using ultrasonography at the start of the experiment, weaning, and day 201. Feeding high vs. low amounts of MA increased the preweaning growth in heifer calves (P = 0.02) without negatively affecting postweaning average daily gain (ADG) (P = 0.74). Compared with heifers fed with low MA, high MA fed heifers had a greater increase in antibodies against Leptospira and Clostridia by 13 wk of age (P = 0.0007 and P = 0.06, respectively). By 27 wk of age, the antibody response was the same in heifers offered low or high MA. There was no effect of MA on the total size of the mammary gland, measured by ultrasonography, at weaning and 7 mo of age. However, the greater MA was associated with more mammary parenchyma (P = 0.01) and less mammary fat pad (P = 0.03) in back glands at 7 mo of age compared with heifers fed lower MA. In conclusion, feeding a high vs. a low amount of unpasteurized whole milk increased the preweaning growth of New Zealand replacement heifers without negatively affecting their ADG during postweaning under grazing conditions. Feeding more (8 vs. 4 L·d-1) unpasteurized whole milk positively affected antibody responses early in life and mammary gland composition by 7 mo of age in dairy heifers reared for pasture-based dairy systems.

This study evaluated the effect of unpasteurized whole milk allowance on intake, antibody response, mammary gland growth, and growth performance of heifers until 7 mo of age. Feeding greater (8 L·d−1) vs. lower (4 L·d−1) milk allowance to heifer calves increased preweaning body weight without having any negative effect on postweaning growth under grazing. Heifers fed high milk allowance had significantly better antibody responses against Leptospira and Clostridia by 3 mo of age and had more mammary parenchyma (potential milk making tissue), and less mammary fat pad (supporting tissue) by 7 mo of age.

Mapping immunogenic epitopes of an adhesin-like protein from Methanobrevibacter ruminantium M1 and comparison of empirical data with in silico prediction methods.

In silico prediction of epitopes is a potentially time-saving alternative to experimental epitope identification but is often subject to misidentification of epitopes and may not be useful for proteins from archaeal microorganisms. In this study, we mapped B- and T-cell epitopes of a model antigen from the methanogen Methanobrevibacter ruminantium M1, the Big_1 domain (AdLP-D1, amino acids 19-198) of an adhesin-like protein. A series of 17 overlapping 20-mer peptides was selected to cover the Big_1 domain. Peptide-specific antibodies were produced in mice and measured by ELISA, while an in vitro splenocyte re-stimulation assay determined specific T-cell responses. Overall, five peptides of the 17 peptides were shown to be major immunogenic epitopes of AdLP-D1. These immunogenic regions were examined for their localization in a homology-based model of AdLP-D1. Validated epitopes were found in the outside region of the protein, with loop like secondary structures reflecting their flexibility. The empirical data were compared with epitope predictions made by programmes based on a range of algorithms. In general, the epitopes identified by in silico predictions were not comparable to those determined empirically.

Mycobacterium avium subsp. paratuberculosis antigens induce cellular immune responses in cattle without causing reactivity to tuberculin in the tuberculosis skin test.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhea, weight-loss, and eventual death in ruminants. Commercially available vaccine provides only partial protection against MAP infection and can interfere with the use of current diagnostic tests for bovine tuberculosis in cattle. Here, we characterized immune responses in calves to vaccines containing four truncated MAP antigens as a fusion (Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786), either displayed on protein particles, or expressed as a soluble recombinant MAP (rMAP) fusion protein as well as to commercially available Silirum® vaccine. The rMAP fusion protein elicited the strongest antigen-specific antibody responses to both PPDA and recombinant antigen and strong and long-lasting T-cell immune responses to these antigens, as indicated by increased production of IFN-γ and IL-17A in antigen-stimulated whole blood cultures. The MAP fusion protein particle vaccine induced minimal antibody responses and weak IFN-γ responses but stimulated IL-17A responses to recombinant antigen. The immune response profile of Silirum® vaccine was characterized by weak antibodies and strong IFN-γ and IL-17A responses to PPDA. Transcription analysis on antigen-stimulated leukocytes from cattle vaccinated with rMAP fusion protein showed differential expression of several immune response genes and genes involved in costimulatory signaling, TLR4, TLR2, PTX3, PTGS2, PD-L1, IL1B, IL2, IL6, IL12B, IL17A, IL22, IFNG, CD40, and CD86. Moreover, the expression of several genes of immune pathways correlated with cellular immune responses in the rMAP fusion protein vaccinated group. These genes have key roles in pathways of mycobacterial immunity, including autophagy, manipulation of macrophage-mediated killing, Th17- and regulatory T cells- (Treg) mediated responses. Calves vaccinated with either the rMAP fusion protein or MAP fusion protein particle vaccine did not induce reactivity to PPDA and PPDB in a comparative cervical skin test, whereas Silirum® induced reactivity to these tuberculins in most of the vaccinated animals. Overall, our results suggest that a combination of recombinant MAP antigens in the form of a soluble fusion protein vaccine are capable of inducing strong antigen-specific humoral and a balanced Th1/Th17-cell immune response. These findings, together with the absence of reactivity to tuberculin, suggest this subunit vaccine could provide protective immunity against intracellular MAP infection in cattle without compromising the use of current bovine tuberculosis surveillance test.

Characterization of local and peripheral immune system in pregnant and nonpregnant ewes.

Modulation of the immune system is known to be important for successful pregnancy but how immune function might differ between the lymph nodes draining the reproductive tract and peripheral lymph nodes is not well understood. Additionally, if immune system changes in response to the presence of an embryo during early pregnancy, and if this response differs in local versus peripheral immune tissue, has not been well characterized. To address these questions, we examined expression of genes important for immune function using NanoString technology in the ampulla and isthmus of the oviduct, endometrium, lymph nodes draining the reproductive tract (lumbo-aortic and medial iliac) as well as a peripheral lymph node (axillary), the spleen, and circulating immune cells from ewes on day 5 of the estrous cycle or pregnancy. Concentrations of estradiol and progesterone in plasma were also determined. Principal component analysis revealed separation of the local from the peripheral lymph nodes (MANOVA P = 3.245e-08, R2 = 0.3) as well as separation of tissues from pregnant and nonpregnant animals [lymph nodes (MANOVA P = 2.337e-09, R2 = 0.5), reproductive tissues (MANOVA P = 2.417e-14, R2 = 0.47)]. Nine genes were differentially (FDR < 0.10) expressed between lymph node types, with clear difference in expression of these genes between the lumbo-aortic and axillary lymph nodes. Expression of these genes in the medial iliac lymph node was not consistently different to either the axillary or the lumbo-aortic lymph node. Expression of IL10RB was increased (FDR < 0.05) by 24% in the reproductive tissue of the pregnant animals compared to nonpregnant animals. Analysis of gene categories revealed that expression of genes of the T-cell receptor pathway in reproductive tract tissues was associated (P < 0.05) with pregnancy status. In conclusion, assessment of gene expression of reproductive and immune tissue provides evidence for a specialization of the local immune system around the reproductive tract potentially important for successful establishment of pregnancy. Additionally, differences in gene expression patterns in reproductive tissue from pregnant and nonpregnant animals could be discerned as early as day 5 of pregnancy. This was found to be associated with expression of genes important for T-cell function and thus highlights the important role of these cells in early pregnancy.

Strigolactones regulate sepal senescence in Arabidopsis.

Flower sepals are critical for flower development and vary greatly in life span depending on their function post-pollination. Very little is known about what controls sepal longevity. Using a sepal senescence mutant screen, we identified two Arabidopsis mutants with delayed senescence directly connecting strigolactones with senescence regulation in a novel floral context that hitherto has not been explored. The mutations were in the strigolactone biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) and in the strigolactone receptor gene DWARF14 (AtD14). The mutation in AtD14 changed the catalytic Ser97 to Phe in the enzyme active site, which is the first mutation of its kind in planta. The lesion in MAX1 was in the haem-iron ligand signature of the cytochrome P450 protein, converting the highly conserved Gly469 to Arg, which was shown in a transient expression assay to substantially inhibit the activity of MAX1. The two mutations highlighted the importance of strigolactone activity for driving to completion senescence initiated both developmentally and in response to carbon-limiting stress, as has been found for the more well-known senescence-associated regulators ethylene and abscisic acid. Analysis of transcript abundance in excised inflorescences during an extended night suggested an intricate relationship among sugar starvation, senescence, and strigolactone biosynthesis and signalling.

Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections.

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

Self-assembled particulate vaccine elicits strong immune responses and reduces Mycobacterium avium subsp. paratuberculosis infection in mice.

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic progressive granulomatous enteritis leading to diarrhoea, weight loss, and eventual death in ruminants. Commercially available vaccines provide only partial protection against MAP infection and can compromise the use of bovine tuberculosis diagnostic tests. Here, we report the development of a protein-particle-based vaccine containing MAP antigens Ag85A202-347-SOD1-72-Ag85B173-330-74F1-148+669-786 as a fusion ('MAP fusion protein particle'). The fusion antigen displayed on protein particles was identified using mass spectrometry. Surface exposure and accessibility of the fusion antigen was confirmed by flow cytometry and ELISA. The MAP fusion protein particle vaccine induced strong antigen-specific T-cell immune responses in mice, as indicated by increased cytokine (IFN-γ and IL-17A) and costimulatory signals (CD40 and CD86) in these animals. Following MAP-challenge, a significant reduction in bacterial burden was observed in multiple organs of the mice vaccinated with the MAP fusion protein particle vaccine compared with the PBS group. The reduction in severity of MAP infection conferred by the MAP fusion protein particle vaccine was similar to that of Silirum and recombinant protein vaccines. Overall, the results provide evidence that MAP antigens can be engineered as a protein particulate vaccine capable of inducing immunity against MAP infection. This utility offers an attractive platform for production of low-cost particulate vaccines against other intracellular pathogens.

Short Communication: Oral Administration of Heat-killed Lactobacillus brevis KB290 in Combination with Retinoic Acid Provides Protection against Influenza Virus Infection in Mice.

Influenza virus type A (IAV) is a seasonal acute respiratory disease virus with severe symptoms, and an effective preventive measure is required. Despite many reports describing the potentially protective effects of lactic acid bacteria, few studies have investigated the effects of nutritional supplement combinations. This study reports the effect of the combined intake of heat-killed Lactobacillus brevis KB290 (KB290) and vitamin A (VA) on mice challenged with a sublethal dose of IAV. For 2 weeks, five groups of mice were fed either placebo, KB290, VA, or a combination of KB290 and VA (KB290+VA). After subsequent IAV challenge, bodyweight and general health were monitored for up to 2 weeks. Viral titres were determined in the lungs of animal subgroups euthanised at days 3, 7, and 14 after IAV challenge. A significant loss was observed in the bodyweights of IAV-infected animals from day 1 post-IAV challenge, whereas the mice fed KB290+VA did not lose any weight after IAV infection, indicating successful protection from the infection. Additionally, mice in the KB290+VA group showed the highest reduction in lung viral titres. In conclusion, the combination of KB290 and VA could be a useful food supplement relevant for protection against seasonal influenza virus infection in humans.

Differential Transcription of Selected Cytokine and Neuroactive Ligand-receptor Genes in Peripheral Leukocytes from Calves in Response to Cautery Disbudding.

Calf disbudding is a painful husbandry practice on dairy and beef cattle farms. An objective measurement of pain is useful to reliably evaluate the pain intensity and anti-nociceptive (analgesic) efficacy of therapeutic agents. The aim of this study was to investigate the changes in peripheral leucocyte inflammatory cytokine gene expression in calves after disbudding, and to assess whether the changes in cytokine gene expression could be an indicator of the efficacy of analgesic drugs. In a randomised controlled study, 16 calves (aged 31 to 41 days and weighing 58 to 73 kg), undergoing routine disbudding, were randomly allocated into two groups (n = 8 in each group). Calves in the control group received no analgesic, while those in the treatment group received 0.5 mg kg-1 meloxicam subcutaneously prior to disbudding. Disbudding was performed using an electric debudder. Blood (10 mL) was sampled from the jugular vein just before and 4 and 24 h post-disbudding, RNA was extracted from leukocytes, and the transcription of 12 genes of interest was assessed using nCounter gene expression assay. The results showed significantly higher transcription (compared to baseline values) of the studied genes (except CRH, IFNγ, and IL10) in the control group calves at either 4 or 24 h post-disbudding. The administration of meloxicam one hour before disbudding significantly attenuated the upregulation of IL6, PGHS2, TAC1, NOS1, and CRH gene transcription post-disbudding, while it did not suppress the elevated transcription of acute and pro-inflammatory cytokines such as IL1ß, IFNγ, IL8, and TNFα genes. In conclusion, nCounter gene expression assay seems to be a promising tool to study the expression of cytokine genes and thus could be used for the pre-clinical evaluation of novel analgesics.

Lactoferrin concentration and expression in New Zealand cows milked once or twice a day.

This study evaluated the concentration and expression of lactoferrin (LF) in cows selected for once a day (OAD) milking compared to twice a day (TAD) milking. Milk samples were collected from the Massey University TAD and OAD herds. Milk traits and expression of LF and insulin-like growth factor 1 (IGF-1) were analyzed with a general linear model that included the fixed effects of milking frequency, lactation number, interaction between milking frequency and lactation number, and as covariates proportion of F, heterosis F × J and deviation from the herd median calving date. Cows milked OAD produced milk with higher (p < .01) concentrations of protein and lactose than TAD milked cows. Compared to TAD cows, cows milked OAD had higher expression of the LF gene (1.40 vs. 1.29 folds, p = .03) and the IGF-1 gene (1.69 vs. 1.48 folds, p = .007). The correlation between the expression of LF gene and the concentration of LF in milk was strong (r = .66 p < .001), but the correlation between the expression of the IGF-1 gene and LF concentration was stronger (r = .94, p < .001). These results suggest that milking frequency affects the milk composition and expression of milk composition genes at early lactation.

Effect of an injectable trace mineral supplement on the immune response of dairy calves.

On a spring calving, pastoral dairy farm, the first 40 heifer calves born after calving mid-point (50% of the herd calved) were blood sampled within 24 h. Thirty were selected, using stratified randomisation to form two equal groups (treatment and control) with the same distribution of serum total protein, copper, selenium, zinc, and manganese concentrations, age and breed. From the remaining 10 calves, five were randomly selected into a sentinel group to assess field exposure to Salmonella spp. All calves received two injections of a killed vaccine containing Salmonella spp. antigens at 2 and 6 weeks of age. Concurrently, the treatment group were injected with 1 mL/50 kg trace mineral supplement (TMS) containing 40 mg zinc, 10 mg manganese, 5 mg selenium, 15 mg copper per mL. Sentinel animals received no injections. All animals were bled from 2 to 9 weeks for assay of immune function. At three and four weeks, white blood cells from TMS calves had an increased percentage of cells phagocytosing (effect size = 9.36 and 4.35) and increased number of bacteria ingested per cell (effect size = 0.93 and 1.52). No differences were detected in gamma interferon response (effect size <0.15) or Salmonella sp. antibody titres (effect size <0.20).

Impact of early weaning on small intestine, metabolic, immune and endocrine system development, growth and body composition in artificially reared lambs.

AbstractThis study evaluated the effect of early weaning (EW) of artificially reared lambs using a restricted milk replacer (MR) feeding and step-down weaning system on the short- and long-term effects on growth, feed intake, selected blood metabolites and hormones, body composition, and small intestine development. Mixed-sex twin-born 2 to 5 d old lambs were randomly allocated to individual pens and fed MR at 20% of initial individual BW in week 1 and 15% in week 2 followed by weaning off MR by the end of week 4 (EW; n = 16) or week 6 (Control; Ctrl, n = 16) using a step-down procedure. Concentrate starter and fiber diets were offered ad libitum to week 9, then gradually removed over a 10-d period. All lambs were managed as a single group on pasture from weeks 6 to 16 of the trial. Feed intake was recorded daily in the first 6 wk, and BWs recorded weekly. At weeks 2, 4, 6, and 8, and pre- and postclostridial vaccination at week 8, blood samples were collected for analysis of selected blood metabolites, IGF-1, and immune function. Body composition was evaluated in eight animals per group at weeks 4 and 16 after euthanasia, and duodenal samples collected for histomorphometric evaluation. Early weaned lambs had lower DM, ME, CP, and NDF intake than Ctrl lambs at 21, 15, 21, and 36 d of rearing, respectively (P < 0.001), driven by lower intakes of MR from day 15 (P < 0.001) as per the experimental design, and lower total DMI of fiber (P = 0.001) from 21 to 42 d of rearing. Lamb BW tended (P = 0.097) to be lower in EW than Ctrl lambs from 5 to 10 wk of rearing, with lower ADG in EW lambs from weeks 3 to 6 (P = 0.041). Early weaning had negligible effects on duodenal morphology, organ, and carcass weights at weeks 4 and 16. Plasma metabolites (urea nitrogen, triglycerides, NEFA, glucose, and total protein) were similar between groups, while ß-hydroxybutyrate was greater in EW than Ctrl lambs at weeks 4 and 6 (P = 0.018) but not week 8 indicative of early rumen development. Serum IGF-1 tended to be lower in EW than Ctrl lambs from weeks 2 to 6 only (P = 0.065). All lambs developed antibody responses postvaccination and there was no effect of treatment (P = 0.528). The results of this study illustrate that artificially reared lambs can be weaned off MR by 4 or 6 wk of rearing without compromising growth, small intestine morphology, major organ development, and body composition, nor immune function at either 4 (preweaning) or 16 (postweaning) wk of age.

Metabolic and Immunological Effects of Intermittent Fasting on a Ketogenic Diet Containing Medium-Chain Triglycerides in Healthy Dogs.

In several species, intermittent fasting (IF) has been shown to have beneficial effects, including delayed aging, increased lifespan, increased insulin sensitivity, reduced ischemic tissue damage, delayed onset of neurodegenerative disease and improved neuronal repair following injury. However, the metabolic and immunological effects of IF have not been well-established in dogs. The aim of this study was to examine the effects of a 48 h IF regimen using a low fat and a high fat diet in healthy dogs by quantifying the metabolic, hormonal, and immunological changes. We hypothesized that IF dogs would have higher blood ketone and ghrelin concentrations, lower blood leptin, insulin and glucose concentrations, and signs of immunosuppression compared to dogs eating daily. Ten healthy adult dogs were randomized into three group and underwent three feeding regimes in a 3 × 3 Latin square design: twice a day feeding on a low fat (23% energy from fat; LF) diet, 48 h fasting on a low fat diet, and 48 h fasting on a high fat enriched with medium-chain triglycerides (68% energy from fat; HF) diet. Body weight, food intake, activity, blood glucose, ß-hydroxybutyrate, leptin, ghrelin, and insulin were measured. Lymphocyte proliferation and neutrophil/macrophage phagocytosis and respiratory burst were measured as markers of immune function. Nuclear magnetic resonance spectroscopy was used to relatively quantify plasma metabolites. When the dogs were IF on a HF diet, they had the highest concentration of blood ketones (mean 0.061 mmol/L, SD 0.024), whereas they had the lowest concentration (mean 0.018 mmol/L, SD 0.004) when fed daily. Blood glucose and insulin concentrations were lower in IF dogs on a HF diet compared to daily feeding or IF on a LF diet. There was an increase in plasma ß-hydroxybutyrate concentrations, and a reduction in glucose and insulin concentrations when dogs were IF on a HF diet. There was only a decline in the immune parameters studied when the dogs were IF on a LF diet, which was not seen when on the HF diet. The results of this study indicate the potential of IF to be further investigated as a potential beneficial feeding regime for dogs.

Far-off and close-up feeding levels affect immunological performance in grazing dairy cows during the transition period.

During the peripartum period, dairy cows often have signs of inflammation. Various stresses, including infectious and metabolic diseases, have been discussed as causative for this inflammation. In this study, expression profiles for 17 immune markers were measured in whole blood preparations from 78 dairy cows over a time frame starting 1 wk before calving to 4 wk after calving. Additionally, the effects of far-off and close-up feeding on immune function of dairy cows during the peripartum period were investigated. Cows were assigned to 1 of 2 feeding levels in late lactation to achieve a low and high BCS at the time of dry-off (approximately 4.25 and 5.0 on a 10-point scale). Following dry-off, both herds were managed to achieve a BCS of 5.0 one month before calving; this involved controlled feeding (i.e., maintenance) and over-feeding of ME during the far-off dry period. Within each far-off feeding-level treatment, cows were offered 65, 90, or 120% of their precalving ME requirements for 3 wk precalving in a 2 × 3 factorial arrangement. Analysis of gene expression profiles from blood cells revealed effects of time indicating that the transition cow's immune system counteracts the peripartum inflammation, whereas later postcalving it becomes activated to provide protection against postpartum infections. Far-off feeding affected (P < 0.05) the expression of 2 of the investigated genes at calving. Interleukin-6 (IL-6) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in unstimulated, peripheral leukocytes were lower (P < 0.05) in animals from the Far-Off_Over-fed group compared with the Far-Off_Control-fed group. Close-up feeding had several effects on gene expression, indicating that immune function in Feed120 animals was distinct from the Feed90 and Feed65. In conclusion, feeding management precalving becomes an important intervention to ensure immunocompetence at and after calving.

Detection of microRNA in cattle serum and their potential use to diagnose severity of Johne's disease.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, which is characterized by chronic progressive granulomatous enteritis. The infection leads to wasting and weight loss in the animals and eventually death, causing considerable production losses to the agricultural industry worldwide. Currently available ELISA- and PCR-based diagnostic tests have limited sensitivity and specificity during early MAP infection in cattle, suggesting that there is an urgent demand for alternative diagnostic tests. Circulating microRNA (miRNA) have recently gained attention as potential biomarkers for several diseases in humans. However, knowledge and use of miRNA as biomarkers in diseases of ruminants, including Johne's disease, are very limited. Here we used NanoString nCounter technology (NanoString, Seattle, WA), a digital platform for amplification-free and hybridization-based quantitative measurement of miRNA in the sera of noninfected and naturally MAP-infected cattle with different severity of infection. Using probes developed against human miRNA, 26 miRNA were detected in cattle serum; 13 of these miRNA were previously uncharacterized for cattle. Canonical discrimination analysis using 20 miRNA grouped animals into 4 distinct clusters based on their disease status, suggesting that the levels of these miRNA can reflect disease severity. A model was developed using a combination of 4 miRNA (miR-1976, miR-873-3p, miR-520f-3p, and miR-126-3p), which distinguished moderate and severely infected animals from noninfected animals. Our study demonstrated the ability of the NanoString nCounter technology to detect differential expression of circulating miRNA in cattle and contributes to widely growing evidence that miRNA can be used as biomarkers in infectious diseases in cattle.